Homocysteine (Hcy) is a thiol-containing amino acid formed from methionine during S-adenosylmethionine-dependent transmethylation reactions. Intracellular Hcy is remethylated to methionine, or is irreversibly catabolized in a series of reactions to form cysteine. Intracellular Hcy is exported into extracellular fluids such as blood and urine, and circulates mostly in oxidized form, and mainly bound to plasma protein (Refsum, et al., Annu. Rev. Medicine, 49:31–62 (1998)). The amount of Hcy in plasma and urine reflects the balance between Hcy production and utilization. This balance may be perturbed by clinical states characterized by genetic disorders of enzymes involved in Hcy transsulfuration and remethylation (e.g., cystathionine β-synthase and N5,10-methylenetetrahydrofolate reductase or dietary deficiency of vitamins (e.g., vitamin B6, B12 and folate) involved in Hcy metabolism (Baual, et al., Cleveland Clinic Journal of Medicine, 64:543–549 (1997)). In addition, plasma Hcy levels may also be perturbed by some medications such as anti-folate drugs (e.g., methotrexate) used for treatments of cancer or arthritis (Foody, et al., Clinician Reviews, 8:203–210 (1998)).
Severe cases of homocysteinemia are caused by homozygous defects in genes encoding for enzymes involved in Hcy metabolisms. In such cases, a defect in an enzyme involved in either Hcy remethylation or transsulfuration leads to as much as 50-fold elevations of Hcy in the blood and urine. The classic form of such a disorder, congenital homocysteinemia (Hcyemia), is caused by homozygous defects in the gene encoding cystathionine β-synthase (CBS). These individuals suffer from thromboembolic complications at an early age, which result in stroke, myocardial infarction, renovascular hypertension, intermittent claudication, mesenteric ischemic, and pulmonary embolism. Such patients may also exhibit mental retardation and other abnormalities resembling ectopia lentis and skeletal deformities (Perry T., Homocysteine: Selected aspects in Nyham W. L. ed. Heritable disorders of amino acid metabolism. New York, John Wiley & Sons, pp. 419–451 (1974)). It is also known that elevated Hcy levels in pregnant women is related to birth defects of children with neurotube closures (Scott, et al., “The etiology of neural tube defects” in Graham, I., Refsum, H., Rosenberg, I. H., and Ureland P. M. ed. “Homocysteine metabolism: from basic science to clinical medicine” Kluwer Academic Publishers, Boston, pp. 133–136 (1995)). Thus, the diagnostic utility of Hcy determinations has been well documented in these clinical conditions.
It has been demonstrated that even mild or moderately elevated levels of Hcy also increase the risk of atherosclerosis of the coronary, cerebral and peripheral arteries and cardiovascular disease (Boushey, et al., JAMA, 274:1049–1057 (1995)). The prevalence of Hcyemia was shown to be 42%, 28%, and 30% among patients with cerebral vascular disease, peripheral vascular disease and cardiovascular disease, respectively (Moghadasian, et al., Arch. Intern. Med., 157:2299–2307 (1997)). A meta-analysis of 27 clinical studies calculated that each increase of 5 μM in Hcy level increases the risk for coronary artery disease by 60% in men and by 80% in women, which is equivalent to an increase of 20 mg/dl−1 (0.5 mmol/dl−1) in plasma cholesterol, suggesting that Hcy, as a risk factor, is as strong as cholesterol in the general population. Results from these clinical studies concluded that hyperhomocysteinemia is an emerging new independent risk factor for cardiovascular disease, and may be accountable for half of all cardiovascular patients who do not have any of the established cardiovascular risk factors (e.g., hypertension, hypercholesterolemia, cigarette smoking, diabetes mellitus, marked obesity and physical inactivity).
Mild homocysteinemia is mainly caused by heterozygosity of enzyme defects. A common polymorphism in the gene for methylenetetrahydrofolate reductase appears to influence the sensitivity of homocysteine levels to folic acid deficiency (Boers, et al., J. Inher. Metab. Dis., 20:301–306 (1997)). Moreover, plasma homocysteine levels are also significantly increased in heart and renal transplant patients (Ueland, et al., J. Lab. Clin. Med., 114:473–501 (1989)), Alzheimer patients(Jacobsen, et al., Clin. Chem., 44:2238–2239 (1998)), as well as in patients of non-insulin-dependent diabetes mellitus (Ducloux, et al., Nephrol. Dial. Transplantl, 13:2890–2893 (1998)). The accumulating evidence linking elevated homocysteine with cardiovascular disease has prompted the initiation of double-blind, randomized and placebo controlled multicenter clinical trials to demonstrate the efficacy of lowering plasma Hcy in preventing or halting the progress of vascular disease (Diaz-Arrastia, et al., Arch. Neurol., 55:1407–1408 (1998)). Determination of plasma homocysteine levels should be a common clinical practice.
As a risk factor for cardiovascular disease, the determination of total plasma Hcy levels (reduced, oxidized and protein-bound) has been recommended in clinical setting (Hornberger, et al., American J. of Public Health, 88:61–67 (1998)). Since 1982, several methods for determining total plasma Hcy have been described (Mansoor, et al., Anal. BioChem., 200:218–229 (1992); Steir, et al., Arch. Intern. Med. 158:1301–1306 (1998); Ueland, et al., Clin. Chem., 39:1764–1779 ( ) 1993); and Ueland, et al., “Plasma homocysteine and cardiovascular disease” in Francis, R. B. Jr. eds. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, Marcel Dokker, pp. 183–236 (1992); see, also, Ueland, et al., “Plasma homocysteine and cardiovascular disease” in Francis, R. B. Jr. eds. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, Marcel Dokker, pp. 183–236 (1992)). The assay of total Hcy in plasma or serum is complicated by the fact that 70% of plasma Hcy is protein-bound and 20–30% exists as free symmetric or mostly asymmetric mixed disulfides. Free reduced Hcy exists in only trace amounts (Stehouwer, et al., Kidney International, 55308–314 (1999)).
Most of the methods require sophisticated chromatographic techniques such as HPLC, capillary gas chromatography, or mass spectrometry (GC/MS) to directly or indirectly (e.g., enzymatic conversion of Hcy to SAH (S-adenosylhomocysteine) by SAH hydrolase followed by HPLC or TLC separation) measure Hcy. Radioenzymatic conversion of Hcy to radiolabeled SAH by SAH hydrolase prior to TLC separation has also been used. In these assays, chromatographic separation, which is often time-consuming and cumbersome to perform, is a common key step of these methods. More particularly, these methods require highly specialized and sophisticated equipment and well-trained analytic specialists. The use of such equipment is generally not well-accepted in routine clinical laboratory practice.
Immunoassays for Hcy that use a monoclonal antibody against SAH (Araki, et al., J Chromatog., 422:43–52 (1987)) are also known. These assays are based upon conversion of Hcy to SAH, which is then detected by a monoclonal antibody. Monoclonal antibody against albumin-bound Hcy has been developed for determination of albumin-bound Hcy (Stabler, et al., J. Clin. Invest., 81:466–474 (1988)), which is the major fraction of total plasma Hcy. Other immunological protocols are also available (see, e.g., U.S. Pat. Nos. 5,631,127, 5,827,645, 5,958,717, 6,063,581 and 5,885,767). Though immunoassays avoid a time-consuming chromatographic separation step and are amenable to automation, production of monoclonal antibody is expensive, somewhat unpredictable, and often requires secondary or even tertiary antibodies for detection.
It is an object herein to provide assays for quantifying and/or detecting homocysteine in body fluids and body tissues.